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. Author manuscript; available in PMC: 2010 May 1.
Published in final edited form as: Nat Cell Biol. 2009 Sep 27;11(11):1376–1382. doi: 10.1038/ncb1982

Figure 4. Demethylation of H3K9 blocks activation of Tip60 acetyltransferase activity.

Figure 4

(a) Wild type Suv39h1/2+/+ or Suv39h1/2−/− double knockout MEFs were untreated (−) or exposed to bleomycin (5μM for 30 minutes). Tip60 was immunopurified with Tip60 antibody, and associated acetyltransferase activity measured. Mock purification with IgG is shown for comparison. Each data point represents the average of 3 independent assays, results ± SD. (b). Wild type Suv39h1/2+/+ or Suv39h1/2−/− double knockout MEFs were untreated (−) or irradiated (5Gy). Levels of ATM, phospho-ATM (pS1981), histone H3 or H3 trimethylated on lysine 9 (H3K9me3) were monitored by western blot analysis. (c) 293T cells expressing HA-Tip60 were transiently transfected with vector, or expression vectors for the histone demethylases Jmjd3/KDM6B, Jmjd2A/KDM4A or Jmjd2D/KDM4D. Cells were irradiated (5Gy) and immunofluorescent staining used to detect either nbs1 and γH2AX (left hand panel) or FLAG-Tip60 and γH2AX (right hand panel). Scale bar equals 10μM. (d) 293T cells were transiently transfected with vector, Jmjd3/KDM6B, Jmjd2A/KDM4A or Jmjd2D/KDM4D. Exactly 30h post-transfection, cells were exposed to bleomycin (5μM for 30 minutes) as indicated. Tip60 was immunopurified, and the associated acetyltransferase activity measured. Each data point represents the average of 3 independent assays, results ± SD. (e) 293T cells were transiently transfected with vector, Jmjd3/KDM6B, Jmjd2A/KDM4A or Jmjd2D/KDM4D demethylases. Exactly 30h post-transfection, cells were untreated (−) or irradiated (5Gy) and analyzed by western blot analysis for ATM, phospho-ATM (pS1981), chk2 and phospho-chk2 (pT68-chk2), p53 and phospho-p53 (pS15-p53), and H2AX and phospho-H2AX (γH2AX).